| 1. |
- Rivera, L., et al.
(författare)
-
Effective qPCR methodology to quantify the expression of virulence genes in Aeromonas salmonicida subsp salmonicida
- 2015
-
Ingår i: Journal of Applied Microbiology. - : Oxford University Press (OUP). - 1364-5072 .- 1365-2672. ; 118:4, s. 792-802
-
Tidskriftsartikel (refereegranskat)abstract
- Aims This study aimed to select and validate different methodological strategies to quantify the expression of the virulence genes ascC and ascV by qPCR in Aeromonas salmonicida subsp. salmonicida (Aer. salmonicida). Methods and Results Using the geNorm, Normfinder and BestKeeper algorithms, reference genes for the qPCR were selected based on their in vitro expression stabilities in three Aer. salmonicida strains. Gene amplification efficiency was calculated by Real-time PCR Miner and LinReg PCR programmes, which have not been used previously in the analysis of bacterial gene expression. The expression of the ascC and ascV virulence genes in a virulent Aer. salmonicida strain was evaluated by three quantification models, including single (least or most stable) or three most stable reference genes, combined with constant or specific gene amplification efficiency. The most stable reference genes were gyrB, proC and rpoC, while rpoD and fabD were the least stable. Quantification models showed different expression patterns. Conclusions The optimal strategy to quantify mRNA expression was to use a combination of the three algorithms and the quantification model including the three most stable reference genes. Real-time PCR Miner or LinReg PCR were valuable tools to estimate amplification efficiency. Significance and Impact of the Study The methods used in this study gave more reliable expression data using qPCR than previously published methods. The quantification and expression dynamics of virulence genes will contribute to a better understanding of how Aer. salmonicida interacts with its host and the environment, and therefore to the prevention of epizootics due to this pathogen.
|
|
| 2. |
- Akram, Neelam, et al.
(författare)
-
Regulation of proteorhodopsin gene expression by nutrient limitation in the marine bacterium Vibrio sp AND4
- 2013
-
Ingår i: Environmental Microbiology. - : Wiley. - 1462-2912 .- 1462-2920. ; 15:5, s. 1400-1415
-
Tidskriftsartikel (refereegranskat)abstract
- Proteorhodopsin (PR), a ubiquitous membrane photoprotein in marine environments, acts as a light-driven proton pump and can provide energy for bacterial cellular metabolism. However, knowledge of factors that regulate PR gene expression in different bacteria remains strongly limited. Here, experiments with Vibrio sp. AND4 showed that PR phototrophy promoted survival only in cells from stationary phase and not in actively growing cells. PR gene expression was tightly regulated, with very low values in exponential phase, a pronounced peak at the exponential/stationary phase intersection, and a marked decline in stationary phase. Thus, PR gene expression at the entry into stationary phase preceded, and could therefore largely explain, the stationary phase light-induced survival response in AND4. Further experiments revealed nutrient limitation, not light exposure, regulated this differential PR expression. Screening of available marine vibrios showed that the PR gene, and thus the potential for PR phototrophy, is found in at least three different clusters in the genus Vibrio. In an ecological context, our findings suggest that some PR-containing bacteria adapted to the exploitation of nutrient-rich micro-environments rely on a phase of relatively slowly declining resources to mount a cellular response preparing them for adverse conditions dispersed in the water column.
|
|
| 3. |
- Bobay, Benjamin G, et al.
(författare)
-
Chemical shift assignments and secondary structure prediction of the phosphorelay protein VanU from Vibrio anguillarum
- 2014
-
Ingår i: Biomolecular NMR Assignments. - : Springer Netherlands. - 1874-2718 .- 1874-270X. ; 8:1, s. 177-179
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum is a biofilm forming Gram-negative bacterium that survives prolonged periods in seawater and causes vibriosis in marine life. A quorum-sensing signal transduction pathway initiates biofilm formation in response to environmental stresses. The phosphotransferase protein VanU is the focal point of the quorum-sensing pathway and facilitates the regulation between independent phosphorelay systems that activate or repress biofilm formation. Here we report the (1)H, (13)C, and (15)N backbone and side chain resonance assignments and secondary structure prediction for VanU from V. anguillarum.
|
|
| 4. |
- Buchholtz, Christiane, et al.
(författare)
-
Profiling of acylated homoserine lactones of Vibrio anguillarum in vitro and in vivo : Influence of growth conditions and serotype.
- 2006
-
Ingår i: Syst Appl Microbiol. - 0723-2020. ; 29:6, s. 433-45
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum produces several interlinked acylated homoserine lactone (AHL) signal molecules which may influence expression of its virulence factors such as exoprotease production and biofilm formation. Using both thin layer chromatography and HPLC-high resolution mass spectrometry (HPLC-HRMS), we demonstrate in this study that the same types of AHLs are produced by many serotypes of V. anguillarum and that altering in vitro growth conditions (salinity, temperature and iron concentration) has little influence on the AHL-profile. Most strains produced N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL) and N-(3-hydroxy-hexanoyl)-l-homoserine lactone (3-hydroxy-C6-HSL) as the dominant molecules. Also, two spots with AHL activity appeared on TLC plates, which could not be identified as AHL structures. Trace amounts of N-(3-hydroxy-octanoyl)-l-homoserine lactone, N-(3-hydroxy-decanoyl)-l-homoserine lactone and N-(3-hydroxy-dodecanoyl)-l-homoserine lactone (3-hydroxy-C8-HSL, 3-hydroxy-C10-HSL and 3-oxo-C12-HSL, respectively) were also detected by HPLC-HRMS analysis from in vitro cultures. Most studies of quorum sensing (QS) systems have been conducted in vitro, the purpose of our study was to determine if the same acylated homoserine lactones were produced in vivo during infection. Extracts from infected fish were purified using several solid phase extraction strategies to allow chromatographic detection and separation by both TLC and HLPC-HRMS. 3-oxo-C10-HSL and 3-hydroxy-C6-HSL were detected in organs from fish dying from vibriosis, however, compared to in vitro culturing where 3-oxo-C10-HSL is the dominant molecule, 3-hydroxy-C6-HSL was prominent in the infected fish tissues. Hence, the balance between the QS systems may be different during infection compared to in vitro cultures. For future studies of QS systems and the possible specific interference with expression of virulence factors, in vitro cultures should be optimised to reflect the in vivo situation.
|
|
| 5. |
- Cámara, Miguel, et al.
(författare)
-
Quorum sensing in Vibrio cholerae.
- 2002
-
Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1061-4036 .- 1546-1718.
-
Annan publikation (refereegranskat)
|
|
| 6. |
- Croxatto, Antony, et al.
(författare)
-
A distinctive dual-channel quorum-sensing system operates in Vibrio anguillarum
- 2004
-
Ingår i: Molecular Microbiology. - : Wiley-Blackwell. - 0950-382X .- 1365-2958. ; 52:6, s. 1677-1689
-
Tidskriftsartikel (refereegranskat)abstract
- Many bacterial cells communicate using diffusible signal molecules to monitor cell population density via a process termed quorum sensing. In marine Vibrio species, the Vibrio harveyi-type LuxR protein is a key player in a quorum-sensing phosphorelay cascade, which controls the expression of virulence, symbiotic and survival genes. Previously, we characterized Vibrio anguillarum homologues of LuxR (VanT) and LuxMN (VanMN) and, in this study, we have identified homologues of LuxPQ (VanPQ) and LuxOU (VanOU). In contrast to other Vibrio species, vanT was expressed at low cell density and showed no significant induction as the cell number increased. In addition, although the loss of VanO increased vanT expression, the loss of VanU, unexpectedly, decreased it. Both VanN and VanQ were required for repression of vanT even in a vanU mutant, suggesting an alternative route for VanNQ signal transduction other than via VanU. VanT negatively regulated its own expression by binding and repressing the vanT promoter and by binding and activating the vanOU promoter. The signal relay results in a cellular response as expression of the metalloprotease, empA, was altered similar to that of vanT in all the mutants. Consequently, the V. anguillarum quorum-sensing phosphorelay systems work differently from those of V. harveyi and may be used to limit rather than induce vanT expression.
|
|
| 7. |
|
|
| 8. |
- Croxatto, Antony, et al.
(författare)
-
VanT, a Homologue of Vibrio harveyi LuxR, Regulates Serine, Metalloprotease, Pigment, and Biofilm Production in Vibrio anguillarum
- 2002
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 184:6, s. 1617-1629
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum possesses at least two N-acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi. In this study, we have cloned an additional gene of this circuit, vanT, encoding a V. harveyi LuxR-like transcriptional regulator. A V. anguillarum ΔvanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ. Three lacZ fusions (serA::lacZ, hpdA-hgdA::lacZ, and sat-vps73::lacZ) were identified which exhibited decreased expression in the ΔvanT strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production. V. anguillarum strains require an active VanT to produce high levels of an l-tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation. A V. anguillarum ΔvanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms. Hence, a new member of the V. harveyi LuxR transcriptional activator family has been characterized in V. anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production.
|
|
| 9. |
- Croxatto, Antony, et al.
(författare)
-
Vibrio anguillarum colonization of rainbow trout integument requires a DNA locus involved in exopolysaccharide transport and biosynthesis.
- 2007
-
Ingår i: Environmental Microbiology. - : Wiley. - 1462-2912 .- 1462-2920. ; 9:2, s. 370-382
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum, part of the normal flora of the aquatic milieu, causes a fatal haemorrhagic septicaemia in marine fish. In this study, a rainbow trout model was used to characterize the colonization of fish skin by V. anguillarum. Within 5 h after infection, the bacterium penetrated the skin mucosal layer, attached to the scales within 12 h, and formed a biofilm by 24-48 h. Two divergently transcribed putative operons, orf1-wbfD-wbfC-wbfB and wza-wzb-wzc, were shown to play a role in skin colonization and virulence. The first operon encodes proteins of unknown function. The wza-wzb-wzc genes encode a secretin, tyrosine kinase and tyrosine phosphatase, respectively, which are similar to proteins in polysaccharide transport complexes. Compared with the wild type, polar mutations in wza, orf1 and wbfD caused a decrease in exopolysaccharide biosynthesis but not lipopolysaccharide biosynthesis. The wza and orf1 mutants did not attach to fish scales; whereas, the wbfD mutant had a wild-type phenotype. Moreover, the wza and orf1 mutants had decreased exoprotease activity, in particular the extracellular metalloprotease EmpA, as well as mucinase activity suggesting that these mutations also affect exoenzyme secretion. Thus, the exopolysaccharide transport system in V. anguillarum is required for attachment to fish skin, possibly preventing mechanical removal of bacteria via natural sloughing of mucus.
|
|
| 10. |
- Farto, R, et al.
(författare)
-
Colonization of turbot tissues by virulent and avirulent Aeromonas salmonicida subsp. salmonicida strains during infection
- 2011
-
Ingår i: Diseases of Aquatic Organisms. - : Inter Research. - 0177-5103 .- 1616-1580. ; 95, s. 167-173
-
Tidskriftsartikel (refereegranskat)abstract
- Preventing disease outbreaks in cultured turbot Psetta maxima L. caused by Aeromonas salmonicida subsp. salmonicida (ASS) requires a better understanding of how this pathogen colonizes its host. Distribution of 1 virulent and 2 avirulent ASS strains in turbot tissues was investigated during early and late stages of infection following an immersion challenge. To track bacteria within the turbot, the ASS strains were tagged with green fluorescent protein (GFP). Both virulent and avirulent strains colonized the epidermal mucus, gills, and intestine within the first 12 h post challenge, suggesting that these sites may serve as points of entry into turbot. Although the avirulent strains colonized these initial sites in the turbot tissues, they were rarely found in the internal organs and were cleared from the host 4 d post challenge. In contrast, the virulent ASS strain was found in the liver and kidney as early as 12 h post challenge and was found in the muscle tissue at very late stages of infection. The virulent strain persisted in all tested host tissues until death occurred 7 d post challenge, suggesting that ASS must colonize and survive within the turbot tissues for an infection to result in death of the fish. Comparisons of the distribution profiles of both virulent and avirulent strains during early and late stages of an infection in turbot has provided important information on the route and persistence of an ASS infection in this host.
|
|
